Epithelial sodium channels (ENaCs) play a vital role in regulating sodium entry into polarized epithelia, including the kidney. Maintenance of whole body sodium and water balance depends on continuous regulation of ENaC activity by hormones. There is reason to suspect that studies of ENaC regulation may be compromised by several types of artifacts, of which three varieties will be examined in this project. (A) Protocol dependence in the formation of patches may influence the response to hormones. (B) The effects of hormones may depend on baseline ENaC density in the membrane. (C) The effects of hormones may differ in short- or open-circuited tissues. This project will generally examine ENaC function in A6 cells as a model tissue on a more fundamental level than is usually done. The results will help to establish the validity of much published data in the literature, and will guide future studies by providing information on the intrinsic limitations of different techniques used to study ENaCs.